It is widely known that FX is a vitamin K dependent blood coagulation factor. Like the other vitamin K dependent factors, FX possesses a Gla domain consisting of 11 γ-carboxyglutamic acids (hereinafter also referred to as “Gla”) in the amino acid sequence beginning from the N-terminal to the 39th residue (Non-patent reference 1). In vitro, FX is converted into activated Factor X (hereinafter also referred to as “FXa”) by an activated Factor VII (hereinafter also referred to as “FVIIa”) or an activated Factor IX (hereinafter also referred to as “FIXa”). FX is used for the treatment of hemophilia patients with inhibitor where an inhibitor to FVIII or FIX is produced as a consequence of substitution therapy with said FVIII or FIX.
Human FX, in the course of its biosynthesis, is subject to posttranslational modification such as generation of Gla, cleavage of a prepro sequence (the sequence of FX after this cleavage is shown in SEQ ID NO: 1), β-hydroxylation of aspartic acid at position 63 in SEQ ID NO: 1, asparagine-type glycosylation at positions 181 and 191, serine/threonine-type glycosylation at positions 159, 171 and 443, and the like. It is thought that FX, after being synthesized as a single-chain protein, is subject to limited degradation with furin, a signal peptidase, at the cleavage motif Arg-Arg-Lys-Arg at positions 139 to 142 in SEQ ID NO: 1 to thereby secrete a two-chain protein.
For expression of a recombinant FX, the expression as a two-chain protein is the most important. It is known that a recombinant expression from an expression vector to which cDNA (SEQ ID NO: 3) encoding the amino acid sequence of FX (the amino acid sequence of FX including the prepro sequence is shown in SEQ ID NO: 2) is simply ligated results in expressed products, most of which are secreted into culture supernatant as a single-chain protein and which have a low specific activity.
In general, in recombinant factors, their expression level is often the matter. For genetic recombination of Factor X in the present invention, in addition to its expression level, the process for generating a two-chain protein was thought to be a rate-determining (Non-patent reference 2). In Non-patent reference 2, Himmelspach et al. co-expressed FX with furin so as to promote generation of a two-chain protein with as high an expression level of FX as 120 μg/ml or more but with a low activity of 25%.    Non-patent reference 1: Journal of Thrombosis and Haemostasis, 3: 2633-2648 (2005)    Non-patent reference 2: Thrombosis Research 97: 51-67 (2000)